Humanized antibodies that recognize alpha-synuclein

ABSTRACT

The present application discloses humanized 1H7 antibodies. The antibodies bind to human alpha synuclein and can be used for treatment and diagnosis of Lewy body disease.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of U.S. application Ser. No.13/750,983, filed Jan. 25, 2013, which claims priority to U.S.Provisional Patent Application No. 61/591,835, filed Jan. 27, 2012 andU.S. Provisional Patent Application No. 61/711,207, filed Oct. 8, 2012,which are incorporated by reference in their entirety for all purposes.

BACKGROUND

Synucleinopathies, including Lewy body diseases (LBDs) are characterizedby degeneration of the dopaminergic system, motor alterations, cognitiveimpairment, and formation of Lewy bodies (LBs) and/or Lewy neurites.(McKeith et al., Neurology (1996) 47:1113-24). Synucleinopathies includeParkinson's disease (including idiopathic Parkinson's disease), DiffuseLewy Body Disease (DLBD) also known as Dementia with Lewy Bodies (DLB),Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's andParkinson disease, pure autonomic failure and multiple system atrophy(MSA; e.g., Olivopontocerebellar Atrophy, Striatonigral Degeneration andShy-Drager Syndrome). Several nonmotor signs and symptoms are thought tobe harbingers for synucleinopathies in the prodromal phase of thediseases (i.e., the presymptomatic, subclinical, preclinical, orpremotor period). Such early signs include, for example, REM sleepbehavior disorder (RBD), loss of smell and constipation (Mahowald etal., Neurology (2010) 75:488-489). Lewy body diseases continue to be acommon cause for movement disorders and cognitive deterioration in theaging population (Galasko et al., Arch. Neurol. (1994) 51:888-95).

Alpha-synuclein is part of a large family of proteins including beta-and gamma-synuclein and synoretin. Alpha-synuclein is expressed in thenormal state associated with synapses and is believed to play a role inneural plasticity, learning and memory. Several studies have implicatedalpha-synuclein with a central role in PD pathogenesis. The protein canaggregate to form insoluble fibrils in pathological conditions. Forexample, synuclein accumulates in LBs (Spillantini et al., Nature (1997)388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi etal., Neurosci. Lett. (1997) 239:45-8). Mutations in the alpha-synucleingene co-segregate with rare familial forms of parkinsonism (Kruger etal., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997)276:2045-7). Over expression of alpha synuclein in transgenic mice(Masliah et al., Science (2000) 287:1265-9) and Drosophila (Feany etal., Nature (2000) 404:394-8) mimics several pathological aspects ofLewy body disease. In addition, it has been suggested that solubleoligomers of synuclein may be neurotoxic (Conway et al., Proc. Natl.Acad. Sci. USA (2000) 97:571-576; Volles et al., J. Biochemistry (2003)42:7871-7878). The accumulation of alpha-synuclein with similarmorphological and neurological alterations in species and animal modelsas diverse as humans, mice, and flies suggests that this moleculecontributes to the development of Lewy body disease.

SUMMARY OF THE CLAIMED INVENTION

The invention provides antibodies comprising a mature heavy chainvariable region comprising the three Kabat CDRs of SEQ ID NO:44, andbeing at least 90% identical to SEQ ID NO:44, and a light chaincomprising the three Kabat CDRs of SEQ ID NO:45, and being at least 90%identical to SEQ ID NO:45. In some antibodies, the mature heavy chainvariable region is at least 95%, 96%, 97%, 98%, or 99% identical to SEQID NO:44 and mature light chain variable region is at least 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO:45. In some antibodies, positionL46 (Kabat numbering) can be occupied by F; position L49 (Kabatnumbering) can be occupied by C; position L83 (Kabat numbering) can beoccupied by A; position H11 (Kabat numbering) can be occupied by L;position H28 (Kabat numbering) can be occupied by S; position H38 (Kabatnumbering) can be occupied by K; position H48 (Kabat numbering) can beoccupied by I; position H67 (Kabat numbering) can be occupied by A;position H69 (Kabat numbering) can be occupied by L; position H71 (Kabatnumbering) can be occupied by A; and/or position H91 (Kabat numbering)can be occupied by F. In some of such antibodies, position H97 (Kabatnumbering) can be occupied by S. In some of such antibodies the aminoacid sequence of the mature heavy chain variable region is SEQ ID NO:44and the amino acid sequence of the mature light chain variable region isSEQ ID NO:45 except provided that position L46 (Kabat numbering) can beoccupied by L or F and/or position L49 (Kabat numbering) can be occupiedby Y or C and/or position L83 (Kabat numbering) can be occupied by F orA, and/or position H11 (Kabat numbering) can be occupied by V or L,and/or position H28 (Kabat numbering) can be occupied by T or S, and/orposition H38 (Kabat numbering) can be occupied by R or K, and/orposition H48 (Kabat numbering) can be occupied by M or I, and/orposition H67 (Kabat numbering) can be occupied by V or A, and/orposition H69 (Kabat numbering) can be occupied by M or L, and/orposition H71 (Kabat numbering) can be occupied by T or A, and/orposition H91 (Kabat numbering) can be occupied by Y or F, and/or H97(Kabat numbering) can be occupied by Cor S. In some of such antibodies,position H71 (Kabat numbering) is occupied by A. In some of suchantibodies, position H67 (Kabat numbering) is occupied by A, positionH71 (Kabat numbering) is occupied by A. In some of such antibodies,position L46 (Kabat numbering) is occupied by F. In some of suchantibodies, position L46 (Kabat numbering) is occupied by F, positionL49 (Kabat numbering) is occupied by C. In some of such antibodies,position L46 (Kabat numbering) is occupied by F, position L49 (Kabatnumbering) is occupied by Y. In some of such antibodies, position H67(Kabat numbering) is occupied by A, position H71 (Kabat numbering) isoccupied by A, L46 (Kabat numbering) is occupied by F, and position L49(Kabat numbering) is occupied by C. In some of such antibodies, positionH11 (Kabat numbering) is occupied by L and position H38 (Kabatnumbering) is occupied by K. In some of such antibodies, position H11(Kabat numbering) is occupied by V and position H38 (Kabat numbering) isoccupied by R. In some of such antibodies, position H28 (Kabatnumbering) is occupied by S, position H48 (Kabat numbering) is occupiedby I, position H69 (Kabat numbering) is occupied by L, position H91(Kabat numbering) is occupied by F. In some of such antibodies, positionH28 (Kabat numbering) is occupied by T, position H48 (Kabat numbering)is occupied by M, position H69 (Kabat numbering) is occupied by M,position H91 (Kabat numbering) is occupied by Y. In some of suchantibodies, position L83 (Kabat numbering) is occupied by A. In some ofsuch antibodies, position L83 (Kabat numbering) is occupied by F. Insome of such antibodies, position H97 (Kabat numbering) is occupied byS. In some of such antibodies, position H97 (Kabat numbering) isoccupied by C. In some of such antibodies, position L46 (Kabatnumbering) is occupied by F, position L49 (Kabat numbering) is occupiedby C and position L83 (Kabat numbering) is occupied by A. In some ofsuch antibodies, position L46 (Kabat numbering) is occupied by F,position L49 (Kabat numbering) is occupied by Y and position L83 (Kabatnumbering) is occupied by A. In some of such antibodies, position L46(Kabat numbering) is occupied by F, position L49 (Kabat numbering) isoccupied by C and position L83 (Kabat numbering) is occupied by F. Insome of such antibodies, position L46 (Kabat numbering) is occupied byF, position L49 (Kabat numbering) is occupied by Y and position L83(Kabat numbering) is occupied by F. In some of such antibodies, positionH11 (Kabat numbering) is occupied by L, position H28 (Kabat numbering)is occupied by S, position H38 (Kabat numbering) is occupied by K,position H48 (Kabat numbering) is occupied by I, position H67 (Kabatnumbering) is occupied by A, position H69 (Kabat numbering) is occupiedby L, position H71 (Kabat numbering) is occupied by A, position H91(Kabat numbering) is occupied by F and position H97 (Kabat numbering) isoccupied by C. In some of such antibodies, position H11 (Kabatnumbering) is occupied by V, position H28 (Kabat numbering) is occupiedby S, position H38 (Kabat numbering) is occupied by R, position H48(Kabat numbering) is occupied by I, position H67 (Kabat numbering) isoccupied by A, position H69 (Kabat numbering) is occupied by L, positionH71 (Kabat numbering) is occupied by A, position H91 (Kabat numbering)is occupied by F and position H97 (Kabat numbering) is occupied by C. Insome of such antibodies, position H11 (Kabat numbering) is occupied byV, position H28 (Kabat numbering) is occupied by T, position H38 (Kabatnumbering) is occupied by R, position H48 (Kabat numbering) is occupiedby M, position H67 (Kabat numbering) is occupied by A, position H69(Kabat numbering) is occupied by M, position H71 (Kabat numbering) isoccupied by A, position H91 (Kabat numbering) is occupied by Y andposition H97 (Kabat numbering) is occupied by C. In some of suchantibodies, position H11 (Kabat numbering) is occupied by V, positionH28 (Kabat numbering) is occupied by S, position H38 (Kabat numbering)is occupied by R, position H48 (Kabat numbering) is occupied by I,position H67 (Kabat numbering) is occupied by A, position H69 (Kabatnumbering) is occupied by L, position H71 (Kabat numbering) is occupiedby A, position H91 (Kabat numbering) is occupied by F and position H97(Kabat numbering) is occupied by S. In some of such antibodies, positionH11 (Kabat numbering) is occupied by V, position H28 (Kabat numbering)is occupied by T, position H38 (Kabat numbering) is occupied by R,position H48 (Kabat numbering) is occupied by M, position H67 (Kabatnumbering) is occupied by A, position H69 (Kabat numbering) is occupiedby M, position H71 (Kabat numbering) is occupied by A, position H91(Kabat numbering) is occupied by Y and position H97 (Kabat numbering) isoccupied by S. In some of such antibodies the amino acid sequence of themature heavy chain variable region is otherwise that of SEQ ID NO:44 andthe amino acid sequence of the mature light chain variable region isotherwise that of SEQ ID NO:45.

The invention further provides an antibody comprising a humanized heavychain comprising the three Kabat CDRs of SEQ ID NO:44 and a humanizedlight chain comprising the three CDRs of SEQ ID NO:45 provided thatposition L46 (Kabat numbering) is occupied by F and/or position L49(Kabat numbering) is occupied by C and/or position L83 (Kabat numbering)is occupied by F, and/or position H11 (Kabat numbering) is occupied byV, and/or position H28 (Kabat numbering) is occupied by T, and/orposition H38 (Kabat numbering) is occupied by R, and/or position H48(Kabat numbering) is occupied by M, and/or position H67 (Kabatnumbering) is occupied by A, and/or position H69 (Kabat numbering) isoccupied by M, and/or position H71 (Kabat numbering) is occupied by A,and/or position H91 (Kabat numbering) is occupied by Y, and/or positionH97 (Kabat numbering) is occupied by C. In some of such antibodies,position L46 (Kabat numbering) is occupied by F, and position L49 (Kabatnumbering) is occupied by C. In some of such antibodies, position H67(Kabat numbering) is occupied by A, and position H71 (Kabat numbering)is occupied by A. In some of such antibodies, position L46 (Kabatnumbering) is occupied by F, position H67 (Kabat numbering) is occupiedby A, position H71 (Kabat numbering) is occupied by A, and position L49(Kabat numbering) is occupied by C. In some of such antibodies, positionH11 (Kabat numbering) is occupied by V and position H38 (Kabatnumbering) is occupied by R. In some of such antibodies, position H28(Kabat numbering) is occupied by T, position H48 (Kabat numbering) isoccupied by M, position H69 (Kabat numbering) is occupied by M, andposition H91 (Kabat numbering) is occupied by Y. In some of suchantibodies, position L49 (Kabat numbering) is occupied by C and positionL83 (Kabat numbering) is occupied by F. In some of such antibodies,position H28 (Kabat numbering) is occupied by T, position H48 (Kabatnumbering) is occupied by M, position H69 (Kabat numbering) is occupiedby M, position H91 (Kabat numbering) is occupied by Y, position L49(Kabat numbering) is occupied by C, and position L83 (Kabat numbering)is occupied by F. In some of such antibodies, position H97 (Kabatnumbering) is occupied by C. In some of such antibodies, position L46(Kabat numbering) is occupied by F, position L49 (Kabat numbering) isoccupied by C, position L83 (Kabat numbering) is occupied by F, positionH11 (Kabat numbering) is occupied by V, H28 (Kabat numbering) isoccupied by T, position H38 (Kabat numbering) is occupied by R, positionH48 (Kabat numbering) is occupied by M, position H67 (Kabat numbering)is occupied by A, position H69 (Kabat numbering) is occupied by M,position H71 (Kabat numbering) is occupied by A, position H91 (Kabatnumbering) is occupied by Y, and H97 (Kabat numbering) is occupied by C.

The invention further provides an antibody comprising a mature heavychain variable region having an amino acid sequence at least 90%identical to SEQ ID NO:23 and a mature light chain variable region atleast 90% identical to SEQ ID NO:37. In some of such antibodies, themature heavy chain variable region comprises the three Kabat CDRs of SEQID NO:23 and the mature light chain variable region comprising the threeKabat CDRs of SEQ ID NO:37, and position H67 (Kabat numbering) isoccupied by A, H71 (Kabat numbering) is occupied by A, position L46(Kabat numbering) is occupied by F, and position L49 (Kabat numbering)is occupied by C. In some of such antibodies, position L83 (Kabatnumbering) is occupied by F. In some of such antibodies, position H97(Kabat numbering) is occupied by C. In some of such antibodies, themature heavy chain has at least 95% sequence identity to SEQ ID NO:23and the mature light chain has at least 95% sequence identity to SEQ IDNO:37. In some of such antibodies, any differences in CDRs of the matureheavy chain variable region and mature light variable region from SEQ IDNOs. 52 and 60 respectively reside in positions H60-H65. In some of suchantibodies, the K_(D) for alpha-synuclein of the antibody is from about0.5 to 2 of K_(D) for alpha-synuclein of a murine or chimeric 1H7antibody. In some of such antibodies, position L83 (Kabat numbering) isoccupied by F or A, position H11 (Kabat numbering) is occupied by V, H28(Kabat numbering) is occupied by T, position H38 (Kabat numbering) isoccupied by R, position H48 (Kabat numbering) is occupied by M, positionH67 (Kabat numbering) is occupied by A, position H69 (Kabat numbering)is occupied by M, position H71 (Kabat numbering) is occupied by A,position H91 (Kabat numbering) is occupied by Y, and H97 (Kabatnumbering) is occupied by C. In some of such antibodies, position L83(Kabat numbering) is occupied by F. In some of such antibodies, positionH97 (Kabat numbering) is occupied by C. In some of such antibodies, anydifference in the variable region framework of the mature heavy chainvariable region and SEQ ID NO:23 are one or more of position H11 (Kabatnumbering) occupied by V, H28 (Kabat numbering) occupied by T, positionH38 (Kabat numbering) occupied by R, position H48 (Kabat numbering)occupied by M, position H69 (Kabat numbering) occupied by M, andposition H91 (Kabat numbering) occupied by Y. In some of suchantibodies, any difference in the variable region framework of themature light chain variable region and SEQ ID NO:37 is position L83(Kabat numbering) occupied by F or A. In some of such antibodies, themature heavy chain variable region has an amino acid sequence designatedSEQ ID NO:23 and the mature light chain variable region has an aminoacid sequence designated SEQ ID NO:37.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence designated SEQ ID NO:44 and the mature light chainvariable region has an amino acid sequence designated SEQ ID NO:45, 33,35, 37, or 39. In some antibodies, the mature heavy chain variableregion has an amino acid sequence designated SEQ ID NO:19 and the maturelight chain variable region has an amino acid sequence designated SEQ IDNO: 45, 33, 35, 37, or 39. In some antibodies, the mature heavy chainvariable region has an amino acid sequence designated SEQ ID NO:21 andthe mature light chain variable region has an amino acid sequencedesignated SEQ ID NO:45, 33, 35, 37, or 39. In some antibodies, themature heavy chain variable region has an amino acid sequence designatedSEQ ID NO:23 and the mature light chain variable region has an aminoacid sequence designated SEQ ID NO: 45, 33, 35, 37, or 39. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:25 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO: 45, 33, 35, 37,or 39. In some antibodies, the mature heavy chain variable region has anamino acid sequence designated SEQ ID NO:27 and the mature light chainvariable region has an amino acid sequence designated SEQ ID NO: 45, 33,35, 37, or 39.

In any of the above antibodies, the mature heavy chain variable regioncan be fused to a heavy chain constant region and the mature light chainconstant region can be fused to a light chain constant region.

In any of the above antibodies, the heavy chain constant region can be amutant form of natural human constant region which has reduced bindingto an Fcγ receptor relative to the natural human constant region.

In any of the above antibodies, the heavy chain constant region can beof human IgG1 isotype. In some antibodies the allotype is G1 m3. In someantibodies, the allotype is G1m1.

In some antibodies, the heavy chain constant region has the amino acidsequence designated SEQ ID NO:52 provided the C-terminal lysine residuemay be omitted. In some antibodies, the light chain constant region hasthe amino acid sequence designated SEQ ID NO:49. In some antibodies, themature heavy chain variable region is fused to a heavy chain constantregion having the amino acid sequence designated SEQ ID NO:52 providedthe C-terminal lysine residue may be omitted and the mature light chainconstant region is fused to a light chain constant region having theamino acid sequence designated SEQ ID NO:49. In some antibodies, themature light chain comprises SEQ ID NO:53 and the mature heavy chaincomprises SEQ ID NO:56.

The invention further provides a nucleic acid encoding any of theabove-mentioned mature heavy chain variable regions and/or any of theabove-mentioned mature light chain variable region, e.g., SEQ ID NOS:18, 20, 22, 24, 26, 32, 34, 36, or 38.

The invention further provides a host cell comprising a vectorcomprising any of the nucleic acids described above.

The invention further provides a pharmaceutical composition comprisingany of the above-mentioned antibodies.

The invention further provides a method of treating a patient having orat risk of a Lewy body disease, comprising administering to the patientan effective regime of any of the above-mentioned antibodies. In somemethods, the disease is Parkinson's disease. In some methods, decline ofcognitive function in the patient is inhibited. In some methods,neuritic and/or axonal alpha synuclein aggregates are reduced. In somemethods, neuritic dystrophy in the patient is reduced. In some methods,synaptic and/or dendritic density is preserved. In some methods, themethod preserves synaptophysin and/or MAP2 in the patient.

The invention further provides a method of reducing Lewy body formationin a patient having or at risk of a Lewy body disease, comprisingadministering to the patient an effective amount of any of theabove-mentioned antibodies. In some methods, the disease is Parkinson'sdisease. In some methods, decline of cognitive function in the patientis inhibited. In some methods, neuritic and/or axonal alpha synucleinaggregates are reduced. In some methods, neuritic dystrophy in thepatient is reduced. In some methods, synaptic and/or dendritic densityis preserved. In some methods, the method preserves synaptophysin and/orMAP2 in the patient.

The invention further provides a method of inhibiting synucleinaggregation or clearing Lewy bodies or synuclein aggregates in a patienthaving or at risk of a Lewy body disease, comprising administering tothe patient an effective amount of any of the above-mentionedantibodies. In some methods, the disease is Parkinson's disease. In somemethods, decline of cognitive function in the patient is inhibited. Insome methods, neuritic and/or axonal alpha synuclein aggregates arereduced. In some methods, neuritic dystrophy in the patient is reduced.In some methods, synaptic and/or dendritic density is preserved. In somemethods, the method preserves synaptophysin and/or MAP2 in the patient.

The invention further provides methods of detecting Lewy bodies in apatient having or at risk of a Lewy body disease, comprisingadministering to the patient an effective amount of any of theabove-mentioned antibodies, wherein the antibody binds to Lewy bodiesand bound antibody is detected. In some methods, the disease isParkinson's disease. In some methods, the antibody is labeled.

The invention further provides a method of producing an antibody,comprising culturing cells transformed with nucleic acids encoding theheavy and light chains of the antibody, so that the cell secrete theantibody; and purifying the antibody from cell culture media; whereinthe antibody is any of the antibodies described above.

The invention further provides a method producing a cell line producingan antibody, comprising introducing a vector encoding heavy and lightchains of an antibody and a selectable marker into cells; propagatingthe cells under conditions to select for cells having increased copynumber of the vector; isolating single cells from the selected cell; andbanking cells cloned from a single cell selected based on yield ofantibody; wherein the antibody is any of the antibodies described above.Some such methods further comprises propagating the cells underselective conditions and screening for cell lines naturally expressingand secreting at least 100 mg/L/10⁶ cells/24 h.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B shows an alignment of the amino acid sequences of m1H7 withfour versions the humanized 1H7 heavy chain mature variable region.BAC02037 (SEQ ID NO:42) is human acceptor V_(H) sequence. CDR regionsaccording to Kabat definition are underlined and in bold.

FIGS. 2A-B shows an alignment of the amino acid sequences of m1H7 withfour versions the humanized 1H7 light chain mature variable region.AAY33358 (SEQ ID NO:43) is human acceptor V_(L) sequence. CDR regionsaccording to Kabat definition are underlined and in bold.

FIGS. 3A-C shows Biacore binding kinetic analysis of murine 1H7 (A),chimeric 1H7 (B) and humanized 1H7 Hu1H7VHv3-Hu1H7VLv3, respectively.

FIGS. 4A-C shows binding kinetic parameters (ForteBio) humanized 1H7(Hu1H7VHv2-Hu1H7VLv4, Hu1H7VHv3-Hu1H7VLv1, Hu1H7VHv3-Hu1H7VLv2,Hu1H7VHv3-Hu1H7VLv3, Hu1H7VHv3-Hu1H7VLv4, Hu1H7VHv4-Hu1H7VLv1) andchimeric 1H7.

FIGS. 5A-B shows the results of passive immunotherapy with 1H7 on memoryperformance in the Morris water maze test.

FIGS. 6A-B shows the results of passive immunotherapy with 1H7 on speedand errors in the round beam test.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the natural human wildtype alpha-synuclein amino acidsequence.

SEQ ID NO:2 is the non-amyloid component (NAC) domain of alpha-synucleinas reported by Jensen et al. (Biochem. J. 310 (Pt 1): 91-94, 1995;GenBank accession number S56746).

SEQ ID NO:3 is the non-amyloid component (NAC) domain of alpha-synucleinas reported by Uéda et al. (Proc. Natl. Acad. Sci. USA, 90:11282-6,1993).

SEQ ID NO:4 is the murine 1H7 antibody(m1H7) heavy chain variablenucleotide sequence.

SEQ ID NO:5 is the m1H7 heavy chain variable amino acid sequence.

SEQ ID NO:6 is the m1H7 light chain variable nucleotide sequence.

SEQ ID NO:7 is the m1H7 light chain variable amino acid sequence.

SEQ ID NO:8 is the mature m1H7 heavy chain variable nucleotide sequence.

SEQ ID NO:9 is the mature m1H7 heavy chain variable amino acid sequence.

SEQ ID NO:10 is the mature m1H7 light chain variable nucleotidesequence.

SEQ ID NO:11 is the mature m1H7 light chain variable amino acidsequence.

SEQ ID NO:12 is the m1H7 heavy chain CDR1 (Kabat definition).

SEQ ID NO:13 is the m1H7 heavy chain CDR2 (Kabat definition).

SEQ ID NO:14 is the m1H7 heavy chain CDR3 (Kabat definition).

SEQ ID NO:15 the m1H7 light chain CDR1 (Kabat definition).

SEQ ID NO:16 is the m1H7 light chain CDR2 (Kabat definition).

SEQ ID NO:17 is the m1H7 light chain CDR3 (Kabat definition).

SEQ ID NO:18 is the Hu1H7VHv1 nucleic acid sequence.

SEQ ID NO:19 is the Hu1H7VHv1 amino acid sequence.

SEQ ID NO:20 is the Hu1H7VHv2 nucleic acid sequence.

SEQ ID NO:21 is the Hu1H7VHv2 amino acid sequence.

SEQ ID NO:22 is the Hu1H7VHv3 nucleic acid sequence.

SEQ ID NO:23 is the Hu1H7VHv3 amino acid sequence.

SEQ ID NO:24 is the Hu1H7VHv4 nucleic acid sequence.

SEQ ID NO:25 is the Hu1H7VHv4 amino acid sequence.

SEQ ID NO:26 is the Hu1H7VHv5 nucleic acid sequence.

SEQ ID NO:27 is the Hu1H7VHv5 amino acid sequence.

SEQ ID NO:28 is the Hu1H7VH signal peptide nucleic acid sequence.

SEQ ID NO:29 is the Hu1H7VH signal peptide amino acid sequence.

SEQ ID NO:30 is the Hu1H7VH signal peptide nucleic acid sequence.

SEQ ID NO:31 is the Hu1H7VH signal peptide amino acid sequence.

SEQ ID NO:32 is the Hu1H7VLv1 nucleic acid sequence.

SEQ ID NO:33 is the Hu1H7VLv1 amino acid sequence.

SEQ ID NO:34 is the Hu1H7VLv2 nucleic acid sequence.

SEQ ID NO:35 is the Hu1H7VLv2 amino acid sequence.

SEQ ID NO:36 is the Hu1H7VLv3 nucleic acid sequence.

SEQ ID NO:37 is the Hu1H7VLv3 amino acid sequence.

SEQ ID NO:39 is the Hu1H7VLv4 amino acid sequence.

SEQ ID NO:40 is the Hu1H7VL signal peptide nucleic acid sequence.

SEQ ID NO:41 is the Hu1H7VL signal peptide amino acid sequence.

SEQ ID NO:42 is the BAC02037 (GI-21670055) human acceptor used for heavychain framework Amino acid sequence.

SEQ ID NO:43 is the AAY33358 (GI-63102905) human acceptor used for lightchain framework amino acid sequence.

SEQ ID NO:44 is the Hu1H7VH having no backmutation or CDR mutation.

SEQ ID NO:45 is the Hu1H7VL having no backmutation or CDR mutation.

SEQ ID NO:46 is the sequence for Hu1H7VH alternatives.

SEQ ID NO:47 is the sequence for Hu1H7VL alternatives.

SEQ ID NO:48 is the sequence for Hu1H7VH CDR3 alternatives.

SEQ ID NO:49 is the Hu1H7 light chain constant region (with arginine)(common for v1-v4).

SEQ ID NO:50 is the Hu1H7 heavy chain constant region (IgG1; common forv1-v5).

SEQ ID NO:51 is the Hu1H7 light chain constant region (without arginine)(common for v1-v4).

SEQ ID NO:52 is the Hu1H7 heavy chain constant region (G1 m3 allotype).

SEQ ID NO:53 is the Hu1H7 light chain version 3 (variableregion+constant region with arginine).

SEQ ID NO:54 is the Hu1H7 light chain version 3 (variableregion+constant region without arginine).

SEQ ID NO:54 is the Hu1H7 heavy chain version 3 (variableregion+constant region).

SEQ ID NO:56 is the Hu1H7 heavy chain version 3 (variableregion+constant region; G1m3 allotype).

SEQ ID NO:57 is the Hu1H7 heavy chain constant region (IgG2).

SEQ ID NO:58 is the Hu1H7 heavy chain constant region (G1 ml allotype).

Definitions

Monoclonal antibodies are typically provided in isolated form. Thismeans that an antibody is typically at least 50% w/w pure of proteinsand other macromolecules arising from its production or purification butdoes not exclude the possibility that the monoclonal antibody iscombined with an excess of pharmaceutical acceptable carrier(s) or othervehicle intended to facilitate its use. Sometimes monoclonal antibodiesare at least 60%, 70%, 80%, 90%, 95 or 99% w/w pure of proteins andother macromolecules from production or purification.

Specific binding of a monoclonal antibody to its target antigen means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific bindingis detectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces. Specific bindingdoes not however necessarily imply that a monoclonal antibody binds oneand only one target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes variable regionof about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7, incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabatalso provides a widely used numbering convention (Kabat numbering) inwhich corresponding residues between different heavy chains or betweendifferent light chains are assigned the same number (e.g., H83 meansposition 83 by Kabat numbering in the mature heavy chain variableregion; likewise position L36 means position 36 by Kabat numbering inthe mature light chain variable region). Kabat numbering is usedthroughout in referring to positions in the variable region of anantibody unless explicitly stated otherwise.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′)₂, F(ab)c,diabodies, Dabs, nanobodies, and Fv. Fragments can be produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes a bispecificantibody and/or a humanized antibody. A bispecific or bifunctionalantibody is an artificial hybrid antibody having two differentheavy/light chain pairs and two different binding sites (see, e.g.,Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990);Kostelny et al., J. Immunol. 148:1547-53 (1992)). In some bispecificantibodies, the two different heavy/light chain pairs include ahumanized 1H7 heavy chain/light chain pair and a heavy chain/light chainpair specific for a different epitope on alpha synuclein than that boundby 1H7.

In some bispecific antibodies, one heavy chain light chain pair is ahumanized 1H7 antibody as further disclosed below and the heavy lightchain pair is from an antibody that binds to a receptor expressed on theblood brain barrier, such as an insulin receptor, an insulin-like growthfactor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, ora transferrin receptor (Friden et al., PNAS 88:4771-4775, 1991; Fridenet al., Science 259:373-377, 1993). Such a bispecific antibody can betransferred cross the blood brain barrier by receptor-mediatedtranscytosis. Brain uptake of the bispecific antibody can be furtherenhanced by engineering the bi-specific antibody to reduce its affinityto the blood brain barrier receptor. Reduced affinity for the receptorresulted in a broader distribution in the brain (see, e.g., Atwal. etal. Sci. Trans. Med. 3, 84ra43, 2011; Yu et al. Sci. Trans. Med. 3,84ra44, 2011).

Exemplary bispecific antibodies can also be (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include but are not limited to BiTE (Micromet), DART(MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) orDuoBody (based on Fab arm exchange, Genmab).

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined by X-ray crystallography ofthe antibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50%, for example, 75%, 90%or 99% as measured in a competitive binding assay. Antibodies identifiedby competition assay (competing antibodies) include antibodies bindingto the same epitope as the reference antibody and antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

A “patient” includes a human or other mammalian subject that receiveeither prophylactic or therapeutic treatment.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises antibody may contain the antibody alone or incombination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (SEM) of astated value.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the patient. A “sign” refers to objectiveevidence of a disease as observed by a physician.

Statistical significance means p≦0.05.

“Cognitive function” refers to mental processes such as any or all ofattention, memory, producing and understanding language, solvingproblems, and making an interest in one's surroundings and self-care.

“Enhanced cognitive function” or “improved cognitive function” refers toimprovement relative to a baseline, for example, diagnosis or initiationof treatment. “Decline of cognitive function” refers to a decrease infunction relative to such a base line.

In animal model systems such as rat or mouse, cognitive function may bemeasured methods including using a maze in which subjects use spatialinformation (e.g., Morris water maze, Barnes circular maze, elevatedradial arm maze, T maze and others), fear conditioning, activeavoidance, illuminated open-field, dark activity meter, elevatedplus-maze, two-compartment exploratory test or forced swimming test.

In humans, cognitive function can be measured by one or more of severalstandardized tests. Examples of a test or assay for cognitive functionwere described (Ruoppila, 1. and Suutama, T. Scand. J. Soc. Med. Suppl.53, 44-65, 1997) and include standardized psychometric tests (e.g.Wechsler Memory Scale, the Wechsler Adult Intelligence Scale, Raven'sStandard Progressive Matrices, Schaie-Thurstone Adult Mental AbilitiesTest), neuropsychological tests (e.g. Luria-Nebraska), metacognitiveself-evaluations (e.g. Metamemory Questionnaire), visual-spatialscreening tests (e.g. Poppelreuter's Figures, Clock Recognition,Honeycomb Drawing and Cancellation), cognitive screening tests (e.g.Folstein's Mini Mental State Test) and reaction time tests. Otherstandard tests for cognitive performance include the Alzheimer's DiseaseAssessment Scale-cognitive subscale (ADAS-cog); the clinical globalimpression of change scale (CIBIC-plus scale); the Alzheimer's DiseaseCooperative Study Activities of Daily Living Scale (ADCS-ADL); the MiniMental State Exam (MMSE); the Neuropsychiatric Inventory (NPI); theClinical Dementia Rating Scale (CDR); the Cambridge NeuropsychologicalTest Automated Battery (CANTAB) or the Sandoz ClinicalAssessment—Geriatric (SCAG), Stroop Test, Trail Making, Wechsler DigitSpan, and the CogState computerized cognitive test. In addition,cognitive function may be measured using imaging techniques such asPositron Emission Tomography (PET), functional magnetic resonanceimaging (fMRI), Single Photon Emission Computed Tomography (SPECT), orany other imaging technique that allows one to measure brain function.

DETAILED DESCRIPTION OF THE INVENTION

I. General

The invention provides humanized 1H7 antibodies (Hu1H7 antibodies). Theantibodies are useful for treatment and diagnoses of a Lewy bodydisease.

II. Target Molecules

Natural human wildtype alpha-synuclein is a peptide of 140 amino acidshaving the following amino acid sequence:

(SEQ ID NO: 1) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYVGSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQKTVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (Uéda et al., Proc. Natl. Acad. Sci. USA, 90:11282-6, 1993; GenBankaccession number: P37840). The protein has three recognized domains, aKTKE repeat domain covering amino acids 1-61, a NAC (Non-amyloidcomponent) domain running from about amino acids 60-95, and a C-terminalacidic domain running from about amino acid 98 to 140. Jensen et al.have reported NAC has the amino acid sequence:EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV (SEQ ID NO: 2) (Jensen et al.,Biochem. J. 310 (Pt 1): 91-94, 1995; GenBank accession number S56746).Uéda et al. have reported NAC has the amino acid sequence:KEQVTNVGGAVVTGVTAVAQKTVEGAGS (SEQ ID NO: 3) (Uéda et al., Proc. Natl.Acad. Sci. USA, 90:11282-6, 1993).

Unless otherwise apparent from the context, reference to alpha-synucleinor its fragments includes the natural human wildtype amino acidsequences indicated above, and human allelic variants thereof,particularly those associated with Lewy body disease (e.g., E46K, A30Pand A53T, with the first letter indicates the amino acid in SEQ ID NO:1,the number is the codon position in SEQ ID NO:1, and the second letteris the amino acid in the allelic variant). Such variants can optionallybe present individually or in any combination. The induced mutationsE83Q, A90V, A76T, which enhance alpha synuclein aggregation, can also bepresent individually or in combination with each other and/or humanallelic variants E46K, A30P and A53T.

III. Lewy Body Diseases

Lewy Body Diseases (LBD) are characterized by degeneration of thedopaminergic system, motor alterations, cognitive impairment, andformation of Lewy bodies (LBs). (McKeith et al., Neurology (1996)47:1113-24). Lewy Bodies are spherical protein deposits found in nervecells. Their presence in the brain disrupts the brain's normal functioninterrupting the action of chemical messengers including acetylcholineand dopamine. Lewy Body diseases include Parkinson's disease (includingidiopathic Parkinson's disease), Diffuse Lewy Body Disease (DLBD) alsoknown as Dementia with Lewy Bodies (DLB), Lewy body variant ofAlzheimer's disease (LBV), Combined Alzheimer's and Parkinson diseaseand as multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy,Striatonigral Degeneration and Shy-Drager Syndrome). DLBD sharessymptoms of both Alzheimer's and Parkinson's disease. DLBD differs fromParkinson's disease mainly in the location of Lewy Bodies. In DLBD LewyBodies form mainly in the cortex. In Parkinson's disease, they formmainly in the substantia nigra. Other Lewy Body diseases include PureAutonomic Failure, Lewy body dysphagia, Incidental LBD, and InheritedLBD (e.g., mutations of the alpha-synuclein gene, PARK3 and PARK4).

IV. Antibodies of the Invention

A. Binding Specificity and Functional Properties

Humanized antibodies of the invention specifically bind to human alphasynuclein. The affinity of some humanized antibodies (i.e., Ka) is canbe, for example, within a factor of five or two of that of the mouseantibody (m1H7). Some humanized antibodies have an affinity that is thesame, within SEM, as m1H7. Some humanized antibodies have an affinitygreater than that of mouse 1H7. Preferred humanized antibodies bind tothe same epitope and/or compete with m1H7 for binding to human alphasynuclein.

B. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen et al., U.S. Pat. Nos. 5,530,101and 5,585,089; Winter et al., U.S. Pat. No. 5,225,539; Carter, U.S. Pat.No. 6,407,213; Adair, U.S. Pat. Nos. 5,859,205 and 6,881,557; and Foote,U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be, forexample, a mature human antibody variable region sequence, a compositeof such sequences, a consensus sequence of human antibody variableregion sequences (e.g., light and heavy chain variable region consensussequences of Kabat, 1991, supra), or a germline variable regionsequence.

An example of an acceptor sequence for the heavy chain is the humanmature heavy chain variable region with NCBI accession code BAC02037(GI: 21670055). This acceptor sequence includes two CDRs having the samecanonical form as mouse 1H7 heavy chain and has a sequence identity of65.8% in the heavy chain variable region framework. If a differentacceptor sequence is used, such an acceptor is can be, for example,another mature heavy chain variable region derived from germline VH1-18or a mature heavy chain variable region sequence incorporating one ofthese germ line sequences.

For the light chain, an example of an acceptor sequence is the lightchain mature variable region with NCBI accession code AAY33358(GI:63102905). This acceptor sequence includes two CDRs having the samecanonical form as a mouse 1H7 light chain and has a sequence identity of65.4% in the light chain variable region framework. If a differentacceptor is used, such an acceptor is preferably another mature lightchain sequence derived from the germline A30 or a light chain maturevariable region sequence incorporating one of these germ line sequences.

A humanized antibody of the invention is an antibody having three lightchain and three heavy chain CDRs as defined by Kabat entirely orsubstantially from the donor mouse 1H7 antibody and mature variableregion framework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Likewise a humanized heavychain is a heavy chain having three heavy chain CDRs as defined by Kabatentirely or substantially from the heavy chain of the mouse 1H7antibody, and a mature heavy chain variable sequence and heavy chainconstant region sequence, if present, entirely or substantially fromhuman antibody heavy chain sequence. Likewise a humanized light chain isa light chain having three light chain CDRs as defined by Kabat entirelyor substantially from the light chain of the m1H7 antibody, and a maturelight chain variable sequence and light chain constant region sequence,if present, entirely or substantially from human antibody light chainsequence. A CDR is substantially from m1H7 if at least 60%, 70%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of residues are identical tothe corresponding residues in the corresponding CDR of m1H7. The maturevariable region framework sequences of an antibody chain or the constantregion sequence of an antibody chain are substantially from a humanmature variable region framework sequence or human constant regionsequence respectively when at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% of corresponding residues defined by Kabat are identical.

Certain amino acids from the human mature variable region frameworkresidues can be selected for substitution based on their possibleinfluence on CDR conformation and/or binding to antigen, mediatinginteraction between heavy and light chains, interaction with theconstant region, being a site for desired or undesiredpost-translational modification, being an unusual residue for itsposition in a human variable region sequence and therefore potentiallyimmunogenic, among other reasons. The following 11 variable regionframework positions were considered as candidates for substitutions forone or more of these reasons as further specified in the Examples (L46F,Y49C, F83A, V11L, T28S, R38K, M481, V67A, M69L, T71A, Y91F).

Here as elsewhere, the first-mentioned residue is the residue of ahumanized antibody formed by grafting Kabat CDRs into a human acceptorframework, and the second-mentioned residue is a residue beingconsidered for replacing such residue. Thus within variable regionframeworks, the first mentioned residue is human and within CDRs thefirst mentioned residue is mouse (e.g., C97S).

Amino acid substitutions can be made in the CDRs. One possible variationis to substitute certain residues in the CDRs of the mouse 1H7 antibodywith corresponding residues from human CDRs sequences, typically fromthe CDRs of the human acceptor sequences used in designing theexemplified humanized antibodies. In some antibodies only part of theCDRs, namely the subset of CDR residues required for binding, termed theSDRs, are needed to retain binding in a humanized antibody. CDR residuesnot contacting antigen and not in the SDRs can be identified based onprevious studies (for example residues H60-H65 in CDR H2 are often notrequired), from regions of Kabat CDRs lying outside Chothiahypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecularmodeling and/or empirically, or as described in Gonzales et al., Mol.Immunol. 41:863 (2004). In such humanized antibodies at positions inwhich one or more donor CDR residues is absent or in which an entiredonor CDR is omitted, the amino acid occupying the position can be anamino acid occupying the corresponding position (by Kabat numbering) inthe acceptor antibody sequence. The number of such substitutions ofacceptor for donor amino acids in the CDRs to include reflects a balanceof competing considerations. Such substitutions are potentiallyadvantageous in decreasing the number of mouse amino acids in ahumanized antibody and consequently decreasing potential immunogenicity.However, substitutions can also cause changes of affinity, andsignificant reductions in affinity are preferably avoided. Positions forsubstitution within CDRs and amino acids to substitute can also beselected empirically.

One reason for performing a substitution within a CDR is that a mouseresidue is a site of posttranslational modification that may interferewith expression or assembly of an antibody. Here, position H97 withinCDRH3, which is occupied by a C in mouse 1H7 was identified as a sitefor substitution.

The 11 variable region framework backmutations and 1 CDR substitutionnoted above can be incorporated into humanized 1H7 antibodies in manypermutations. The heavy chain variable region of such antibodies can berepresented by a sequence comprisingQVQLVQSGAE-X₁-KKPGASVKVSCKASGY-X₂-FTSYYIHWV-X₃-QAPGQGLEW-X₄-GWIYPGSGNTKYSEKFKGR-X₅-T-X₆-T-X₇-DTSTSTAYMELRSLRSDDTAVY-X₈-CARDG-X₉-YGFAYWGQGTLVTVSS,wherein -X₁- is V or L; -X₂- is S or T; -X₃- is R or K; -X₄- is M or I;-X₅- is V or A; -X₆- is M or L; -X₇- is T or A; -X₈- is Y or F; -X₉- isC, M, S, or T (SEQ ID NO:46). In some heavy chain variable regions, -X₉-is C. Some light chain variable regions can be represented by a sequencecomprisingDIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPK-Z₁-L₁-Z₂-AASNLESGVPSRFSGSGSGTDFTLTISSLQPED-Z₃-ATYYCQQSNEDPFTFGQGTKLEIK,wherein -Z₁- is L or F; -Z₂- is Y or C; -Z₃- is F or A (SEQ ID NO:47).In some antibodies the heavy chain variable region comprises SEQ ID NO:46 and the light chain variable region comprises SEQ ID NO:47. Forexample, residues, X₁ and X₃ of SEQ ID NO: 48 are V and R, respectively.For example, residues, X₅- and X₇ of SEQ ID NO:46 are A and residue Z₁of SEQ ID NO:47 is F. For example, residues, -X₄, X₆, and X₈ of SEQ IDNO:46 are M, M, and Y, respectively, and residue Z₂ and ₃ of SEQ IDNO:47 are C and F, respectively. For example, residues, X₂ and X₉ of SEQID NO: 48 are T and C, respectively.

Some antibodies contain two heavy chain substitutions and two lightchain substitutions. For example, position H67 is occupied by A, H71 isoccupied by A, position L46 is occupied by F, and position L49 isoccupied by C. In some antibodies, the heavy chain mature variableregion has an amino acid sequence designated SEQ ID NO:23. In someantibodies, the light chain mature variable region has an amino acidsequence designated SEQ ID NO:37. For example, in H3L3 (Hu1H7VHv3 (SEQID NO:23-Hu1H7VLv3 (SEQ ID NO:37)), the heavy chain mature variableregion has an amino acid sequence designated SEQ ID NO:23, and the lightchain mature variable region has an amino acid sequence designated SEQID NO:37.

The invention provides variants of the H3L3 humanized antibody in whichthe humanized heavy chain mature variable region shows at least 90%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:23 and the humanizedlight chain mature variable region shows at least 90%, 95%, 96%, 97%,98% or 99% sequence identity to SEQ ID NO:37. Some such humanizedantibodies include three heavy and three light chain CDRs entirely orsubstantially identical to the CDR regions of H3L3, which are the sameas those of the mouse donor antibody. The CDR regions can be defined byany conventional definition (e.g., Chothia) but are preferably asdefined by Kabat.

Some variants of the H3L3 humanized antibody retain some or all of thebackmutations in H3L3. In other words, at least 1, 2, 3 or preferablyall 4 of the following are present: H67 is occupied by A, and H71 isoccupied by A, L46 is occupied by F, and position L49 is occupied by C.

In addition to retaining at least 1, 2, 3 or preferably all 4 of thebackmutations of H3L3, humanized 1H7 antibodies may also containadditional backmutations in the variable region frameworks. Examples ofsuch backmutations include H11 occupied by L, H28 occupied by S, H38occupied by K, H48 occupied by I, H69 occupied by L, H91 occupied by F,and/or L83 occupied by A. For selection of backmutations for atherapeutic or diagnostic product, one should take into account thedegree to which they in general do not improve affinity and the degreeto which introducing more mouse residues may give increased risk ofimmunogenicity. For example, H3L1 comprises a heavy chain maturevariable region of SEQ ID NO:23, and a light chain of SEQ ID NO:33. Forexample, H4L1 comprises a heavy chain mature variable region of SEQ IDNO:25, and a light chain of SEQ ID NO:33.

Another possibility for variation is to use a different human acceptorsequences as discussed above. Substitutions in CDR regions are possibleas described above, for example at position H97, but prior to selectingsuch substitutions for a therapeutic or diagnostic product, one shouldconsider the potential effect on affinity and antibody expression.

If position H97 in mouse CDRH3 is other than cysteine, it is preferablyoccupied by M, S, or T. Some antibodies comprise a humanized heavy chaincomprising Kabat CDR1 of SEQ ID NO:12: SYYIH; Kabat CDR2 of SEQ IDNO:13: WIYPGSGNTKYSEKFKG; Kabat CDR3 of SEQ ID NO:48: DG-X₉-YGFAY,wherein -X₉- is C, M, S, or T, more preferably C. Some antibodiescomprise a humanized light chain comprising Kabat CDR1 of SEQ ID NO:15:KASQSVDYDGDSYMN; Kabat CDR2 of SEQ ID NO:16: AASNLES; Kabat CDR3 of SEQID NO:17: QQSNEDPFT. Some antibodies comprise a humanized heavy chaincomprising the three Kabat CDRs of SEQ ID NOs:12, 13, and 48 and ahumanized light chain comprising the three Kabat CDRs of SEQ IDNOs:15-17. In some such antibodies, a humanized heavy chain comprisesthe three Kabat CDRs of SEQ ID NO:12-14 and a humanized light chaincomprises the three Kabat CDRs of SEQ ID NO:15-17.

The invention further provides humanized 1H7 antibodies in which thehumanized heavy chain mature variable region shows at least 90%, 95%,96%, 97%, 98%, or 99% identity or is 100% identity with SEQ ID NOs. 19,21, 23, 25, and 27 and the humanized light chain mature variable regionshows at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to oris 100% identical with any one of SEQ ID NOs. 33, 35, 37, and 39.

Various permutations of the 11 variable region framework backmutationsand 1 CDR mutation described above can be incorporated into thehumanized 1H7 antibodies. In some such antibodies, position L46 isoccupied by L or F (preferably F) and/or position L49 is occupied by Yor C (preferably C) and/or position L83 is occupied by F or A, and/orposition H11 is occupied by V or L, and/or position H28 is occupied by Tor S, and/or position H38 is occupied by R or K, and/or position H48 isoccupied by M or I, and/or position H67 is occupied by V or A(preferably A), and/or position H69 is occupied by M or L, and/orposition H71 is occupied by T or A (preferably A), and/or position H91is occupied by Y or F, and/or position H28 is occupied by S or T, and/orposition H97 is occupied by C or M or S or T (preferably C).

In some such antibodies, some or all of the backmutations inHu1H7VLv1-v4 and Hu1H7VHv1-v5 are retained. Preferably, thebackmutations at positions L46, H67, and H71 are retained. In otherwords, position L46 is occupied by F, position H67 is occupied by A, andposition H71 is occupied by A. More preferably, the backmutations atpositions L46, L49, H67, and H71 are retained. In other words, positionL46 is occupied by F, position L49 is occupied by C, position H67 isoccupied by A, and position H71 is occupied by A. In some antibodies,some or all of heavy chain positions H11, H28, H38, H48, H67, H69, H71,and/or H91 are occupied by L, S, K, I, A, L, A, and F respectively.Likewise in some antibodies some or all of light chain positions L46,L49 and/or L83 are occupied by F, C and A respectively. In someantibodies, 1, 2, 3, 4, 5, 6, 7, 8, 9 or all ten of positions H11, H28,H38, H48, H67, H69, H71, H91, L46, L49 and L83 is/are occupied by L, S,K, I, A, L, A, F, F, C and A respectively. In some antibodies, 0, 1, 2,3, 4, 5, 6, or 7 positions are changed in the heavy chain maturevariable region framework relative to SEQ ID NO:44, and 0, 1, 2, or 3positions are change in the light chain mature variable region frameworkrelative to SEQ ID NO:45. In some such antibodies, position H97 isoccupied by C. Preferably, the humanized antibody has a K_(D) foralpha-synuclein from about 0.5 to 2 of that of a murine or chimeric 1H7antibody.

In some antibodies, position L46 is occupied by F, position H67 isoccupied by A, position H71 is occupied by A, position H11 is occupiedby V, and position H38 is occupied by R. In some such antibodies,position L49 is occupied by Y. More preferably, in some such antibodies,position L49 is occupied by C. In some such antibodies, position L83 isoccupied by F. In some such antibodies, position L83 is occupied by A.In some such antibodies, position H97 is occupied by C. In some suchantibodies, position H28 is occupied by T or S, position H48 is occupiedby M, position H69 is occupied by M and position H91 is occupied by Y.In some such antibodies, position H28 is occupied by T or S, positionH48 is occupied by M, position H69 is occupied by M and position H91 isoccupied by F. In some such antibodies, position H28 is occupied by T orS, position H48 is occupied by M, position H69 is occupied by L andposition H91 is occupied by Y. In some such antibodies, position H28 isoccupied by T or S, position H48 is occupied by M, position H69 isoccupied by L and position H91 is occupied by F. In some suchantibodies, position H28 is occupied by T or S, position H48 is occupiedby I, position H69 is occupied by M and position H91 is occupied by Y.In some such antibodies, position H28 is occupied by T or S, positionH48 is occupied by I, position H69 is occupied by M and position H91 isoccupied by F. In some such antibodies, position H28 is occupied by T orS, position H48 is occupied by I, position H69 is occupied by L andposition H91 is occupied by Y. In some such antibodies, position H28 isoccupied by T or S, position H48 is occupied by I, position H69 isoccupied by L and position H91 is occupied by F. In some suchantibodies, position H11 is occupied by L, and/or position H38 isoccupied by K, and/or position H97 is occupied by S.

In any of the above antibodies, other amino acid substitutions can bemade in the mature variable region framework, for example, in residuesnot in contact with the CDRs. Often the replacements made in the varianthumanized sequences are conservative with respect to the replaced aminoacids. In some antibodies, replacements relative to Hu1H7VLv1-v4 andHu1H7VHv1-v5 (whether or not conservative) have no substantial effect onthe binding affinity or potency of the resultant antibody relative toHu1H7VLv1-v4 and Hu1H7VHv1-v5, that is, its ability to bind human alphasynuclein.

Variants typically differ from the heavy and light chain mature variableregion sequences of Hu1H7VLv1-v4 and Hu1H7VHv1-v5 by a small number(e.g., typically no more than 1, 2, 3, 5 or 10 in either the light chainor heavy chain mature variable region framework, or both) ofreplacements, deletions or insertions.

C. Selection of Constant Region

The heavy and light chain variable regions of humanized antibodies canbe linked to at least a portion of a human constant region. The choiceof constant region depends, in part, whether antibody-dependentcell-mediated cytotoxicity, antibody dependent cellular phagocytosisand/or complement dependent cytotoxicity are desired. For example, humanisotopes IgG1 and IgG3 have complement-dependent cytotoxicity and humanisotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce strongercell mediated effector functions than human IgG2 and IgG4. Light chainconstant regions can be lambda or kappa. An exemplary human light chainkappa constant region has the amino acid sequence of SEQ ID NO:49. TheN-terminal arginine of SEQ ID NO:49 can be omitted, in which case lightchain kappa constant region has the amino acid sequence of SEQ ID NO:51.An exemplary human IgG1 heavy chain constant region has the amino acidsequence of SEQ ID NO:50. An exemplary human IgG2 heavy chain constantregion has the amino acid sequence of SEQ ID NO:57. Antibodies can beexpressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′, F(ab′)2, and Fv, oras single chain antibodies in which heavy and light chain maturevariable domains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype bind to a non-polymorphic region of a one or more otherisotypes. Thus, for example, heavy chain constant regions can be of IgG1G1m1 or IgG1 G1 m3 allotypes and have the amino acid sequence of SEQ IDNO:52 or SEQ ID NO:55. Yet another heavy chain constant region has theamino acid sequence of SEQ ID NO:52 or SEQ ID NO:55 except that it lacksthe C-terminal lysine.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reduceaffinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., U.S.Pat. No. 6,624,821). Some antibodies have alanine substitution atpositions 234, 235 and 237 of human IgG1 for reducing effectorfunctions. Optionally, positions 234, 236 and/or 237 in human IgG2 aresubstituted with alanine and position 235 with glutamine (see, e.g.,U.S. Pat. No. 5,624,821).

In some antibodies, the light chain constant region has the amino acidsequence designated SEQ ID NO:49. In some antibodies, the heavy chainconstant region has the amino acid sequence designated SEQ ID NO:52.Exemplary Hu1H7 light chains have SEQ ID NO:53 or SEQ ID NO:54.Exemplary Hu1H7 heavy chains have the amino acid sequence of SEQ IDNO:55 or SEQ ID NO:56. In some antibodies, the light chain is SEQ IDNO:53. In some antibodies, the heavy chain is SEQ ID NO:56.

D. Expression of Recombinant Antibodies

Antibodies can be produced by recombinant expression. Nucleic acidsencoding the antibodies can be codon-optimized for expression in thedesired cell-type (e.g., CHO or Sp2/0). Recombinant nucleic acidconstructs typically include an expression control sequence operablylinked to the coding sequences of antibody chains, includingnaturally-associated or heterologous promoter regions. The expressioncontrol sequences can be eukaryotic promoter systems in vectors capableof transforming or transfecting eukaryotic host cells. Once the vectorhas been incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies. The vector or vectors encoding the antibody chains can alsocontain a selectable gene, such as dihydrofolate reductase, to allowamplification of copy number of the nucleic acids encoding the antibodychains.

E. coli is a prokaryotic host particularly useful for expressingantibodies, particularly antibody fragments. Microbes, such as yeast arealso useful for expression. Saccharomyces is an example of a yeast host,with suitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilizations.

Mammalian cells can be used for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes toClones, (VCH Publishers, NY, 1987). A number of suitable host cell linescapable of secreting intact heterologous proteins have been developed inthe art, and include CHO cell lines, various COS cell lines, HeLa cells,HEK293 cells, L cells, and non-antibody-producing myelomas includingSp2/0 and NS0. It can be advantageous to use nonhuman cells. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter, an enhancer (Queen et al.,Immunol. Rev. 89:49 (1986)), and necessary processing information sites,such as ribosome binding sites, RNA splice sites, polyadenylation sites,and transcriptional terminator sequences. Suitable expression controlsequences are promoters derived from endogenous genes, cytomegalovirus,SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected to FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be advantageous. Antibodies produced by single cell clonescan also be tested for turbidity, filtration properties, PAGE, IEF, UVscan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometery,and binding assay, such as ELISA or Biacore. A selected clone can thenbe banked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, columnchromatography (e.g., hydrophobic interaction or ion exchange), low-pHfor viral inactivation and the like (see generally, Scopes, ProteinPurification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies including codonoptimization, selection of promoters, transcription elements, andterminators, serum-free single cell cloning, cell banking, use ofselection markers for amplification of copy number, CHO terminator,serum free single cell cloning, improvement of protein titers (see,e.g., U.S. Pat. Nos. 5,786,464, 5,888,809, 6,063,598, 6,114,148,7,569,339, WO2004/050884, WO2005/019442, WO2008/012142, WO2008/012142,WO2008/107388, and WO2009/027471, and).

E. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above. Typically, the nucleic acids alsoencode a signal peptide fused to the mature heavy and light chains(e.g., signal peptides having amino acid sequences of SEQ ID NOS: 29, 31and 41 that can be encoded by SEQ ID NOS: 28, 30 and 40). Codingsequences on nucleic acids can be in operable linkage with regulatorysequences to ensure expression of the coding sequences, such as apromoter, enhancer, ribosome binding site, transcription terminationsignal and the like. The nucleic acids encoding heavy and light chainscan occur in isolated form or can be cloned into one or more vectors.The nucleic acids can be synthesized by for example, solid statesynthesis or PCR of overlapping oligonucleotides. Nucleic acids encodingheavy and light chains can be joined as one contiguous nucleic acid,e.g., within an expression vector, or can be separate, e.g., each clonedinto its own expression vector.

V. Therapeutic Applications

The invention provides several methods of treating or effectingprophylaxis of Lewy Body disease in patients suffering from or at riskof such disease. Patients amenable to treatment include individuals atrisk of disease of a LBD but not showing symptoms, as well as patientspresently showing symptoms or the early warning signs ofsynucleinopathies, for example, EEG slowing, neuropsychiatricmanifestations (depression, dementia, hallucinations, anxiety, apathy,anhedonia), autonomic changes (orthostatic hypotension, bladderdisturbances, constipation, fecal incontinence, sialorrhea, dysphagia,sexual dysfunction, changes in cerebral blood flow), sensory changes(olfactory, pain, color discrimination abnormal sensations), sleepdisorders (REM sleep behavior disorder (RBD), restless legssyndrome/periodic extremity movements, hypersomnia, insomnia) andmiscellaneous other signs and symptoms (fatigue, diplopia, blurredvision, seborrhea, weight loss/gain). Therefore, the present methods canbe administered prophylactically to individuals who have a known geneticrisk of a LBD. Such individuals include those having relatives who haveexperienced this disease, and those whose risk is determined by analysisof genetic or biochemical markers. Genetic markers of risk toward PDinclude mutations in the alpha-synuclein or Parkin, UCHLI, and CYP2D6genes; particularly mutations at positions 30 and 53 of thealpha-synuclein gene. Individuals presently suffering from Parkinson'sdisease can be recognized from its clinical manifestations includingresting tremor, muscular rigidity, bradykinesia and posturalinstability.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to a therapeutic agent(e.g., a truncated form of alpha-synuclein peptide) over time. If theresponse falls, a booster dosage is indicated.

Antibodies can be used for treating or effecting prophylaxis of LewyBody disease in patients by administration under conditions thatgenerate a beneficial therapeutic response in a patient (e.g., reductionof neuritic and/or axonal alpha synuclein aggregates, reduction ofneuritic dystrophy, improving cognitive function, and/or reversing,treating or preventing cognitive decline) in the patient. In somemethods, the areas of neuritic dystrophy in the neuropil of neocortexand/or basal ganglia can be reduced by on average at least 10%, 20%,30%, or 40% in treated patients compared with a control population.

Cognitive impairment, progressive decline in cognitive function, changesin brain morphology, and changes in cerebrovascular function arecommonly observed in patients suffering from or at risk of Lewy Bodydisease. Administration of the present antibodies can inhibit or delaydecline of cognitive function in such patients.

The invention also provides methods of preserving or increasing synapticdensity and/or dentritic density. An index of changes in synaptic ordentritic density can be measured by markers of synapse formation(synaptophysin) and/or dendrites (MAP2). In some methods, the synapticor dentritic density can be restored to the level of synaptic ordentritic density in a healthy subject. In some methods, the mean levelof synaptic or dentritic density in treated patients can be elevated by5%, 10%, 15%, 20%, 25%, 30% or more as compared to a population ofuntreated control patients.

VI. Pharmaceutical Compositions and Methods of Treatment

In prophylactic applications, an antibody or agent for inducing anantibody or a pharmaceutical composition the same is administered to apatient susceptible to, or otherwise at risk of a disease in a regime(dose, frequency and route of administration) effective to reduce therisk, lessen the severity, or delay the onset of at least one sign orsymptom of the disease. In some prophylactic applications, the regime iseffective to inhibit or delay accumulation of alpha synuclein andtruncated fragments in the brain, and/or inhibit or delay its toxiceffects and/or inhibit/or delay development of behavioral deficits. Intherapeutic applications, an antibody or agent to induce an antibody isadministered to a patient suspected of, or already suffering from a Lewybody disease in a regime (dose, frequency and route of administration)effective to ameliorate or at least inhibit further deterioration of atleast one sign or symptom of the disease. In some therapeuticapplications, the regime is effective to reduce or at least inhibitfurther increase of levels of alpha synuclein and truncated fragments,associated toxicities and/or behavioral deficits.

A regime is considered therapeutically or prophylactically effective ifan individual treated patient achieves an outcome more favorable thanthe mean outcome in a control population of comparable patients nottreated by methods of the invention, or if a more favorable outcome isdemonstrated in treated patients versus control patients in a controlledclinical trial (e.g., a phase II, phase II/III or phase III trial) atthe p<0.05 or 0.01 or even 0.001 level.

Effective doses vary depending upon many different factors, includingmeans of administration, target site, physiological state of the patientincluding type of Lewy body disease, whether the patient is an ApoEcarrier, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.

An exemplary dosage range for antibodies is from about 0.01 to 5 mg/kg,and more usually 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, ofpatient body weight. Antibody can be administered such doses daily, onalternative days, weekly, fortnightly, monthly, quarterly, or accordingto any other schedule determined by empirical analysis. An exemplarytreatment entails administration in multiple dosages over a prolongedperiod, for example, of at least six months. Additional exemplarytreatment regimes entail administration once per every two weeks or oncea month or once every 3 to 6 months.

Antibodies can be administered via a peripheral route (i.e., one inwhich an administered or induced antibody crosses the blood brainbarrier to reach an intended site in the brain. Routes of administrationinclude topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intrathecal, intraperitoneal, intranasal or intramuscular.Some routes for administration of antibodies are intravenous andsubcutaneous. This type of injection is most typically performed in thearm or leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection.

Pharmaceutical compositions for parenteral administration are can besterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The present regimes can be administered in combination with anotheragent effective in treatment or prophylaxis of the disease beingtreated. For example, in the case of Parkinson's disease, immunotherapyagainst alpha synuclein WO/2008/103472, Levodopa, dopamine agonists,COMT inhibitors, MAO-B inhibitors, Amantadine, or anticholinergic agentscan be used in combination with the present regimes.

VII. Other Applications

The antibodies described above can be used for detecting alpha-synucleinin the context of clinical diagnosis or treatment or in research. Theantibodies can also be sold as research reagents for laboratory researchin detecting cells bearing alpha-synuclein and their response to variousstimuli. In such uses, monoclonal antibodies can be labeled withfluorescent molecules, spin-labeled molecules, enzymes or radioisotypes,and can be provided in the form of kit with all the necessary reagentsto perform the assay for alpha-synuclein. The antibodies can also beused to purify alpha-synuclein, e.g., by affinity chromatography.

The antibodies can be used for detecting LBs in a patient. Such methodsare useful to diagnose or confirm diagnosis of PD, or other diseaseassociated with the presence of LBs in the brain, or susceptibilitythereto. For example, the methods can be used on a patient presentingwith symptoms of dementia. If the patient has LBs, then the patient islikely suffering from a Lewy body disease, such as Parkinson's disease.The methods can also be used on asymptomatic patients. Presence of Lewybodies or other abnormal deposits of alpha-synuclein indicatessusceptibility to future symptomatic disease. The methods are alsouseful for monitoring disease progression and/or response to treatmentin patients who have been previously diagnosed with a Lewy body disease.

The methods can be performed by administering an antibody and thendetecting the antibody after it has bound. If desired, the clearingresponse can be avoided by using an antibody fragment lacking afull-length constant region, such as a Fab. In some methods, the sameantibody can serve as both a treatment and diagnostic reagent.

For diagnosis (e.g., in vivo imaging), the antibodies can beadministered by intravenous injection into the body of the patient, ordirectly into the brain by intracranial injection or by drilling a holethrough the skull. The dosage of reagent should be within the sameranges as for treatment methods. Typically, the antibody is labeled,although in some methods, the antibody is unlabeled and a secondarylabeling agent is used to bind to the antibody. The choice of labeldepends on the means of detection. For example, a fluorescent label issuitable for optical detection. Use of paramagnetic labels is suitablefor tomographic detection without surgical intervention. Radioactivelabels can also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size and/or intensity oflabeled loci to corresponding base line values. The base line values canrepresent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame patient. For example, base line values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line signals a positive response to treatment.

The antibodies can be used to generate anti-idiotype antibodies. (see,e.g., Greenspan & Bona, FASEB J. 7(5):437-444, 1989; and Nissinoff, J.Immunol. 147:2429-2438, 1991). Such anti-idiotype antibodies can beutilized in pharmacokinetics, pharmacodynamics, biodistribution studiesas well as in studies of clinical human-anti-human antibody (HAHA)responses in individuals treated with the antibodies. For example,anti-idiotypic antibodies bind specifically the variable region ofhumanized 1H7 antibodies and therefore can be used to detect humanized1H7 antibodies in pharmacokinetic studies and help to quantifyhuman-anti-human antibody (HAHA) responses in treated individuals.

All patent filings, website, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example I Design of Humanized 1H7 Antibodies

The starting point or donor antibody for humanization is the mouseantibody 1H7 produced by the hybridoma having ATCC Accession No.PTA-8220 and described in U.S. patent application Ser. No. 11/710,248published as US2009/0208487. The complete heavy chain variable aminoacid and nucleic acid sequences of m1H7 are provided as SEQ ID NOS:4 and5, respectively. The complete light chain variable amino acid andnucleic acid sequences of m1H7 are provided as SEQ ID NOS:6 and 7,respectively. The heavy chain variable amino acid and nucleic acidsequences of mature m1H7 are provided as SEQ ID NOS:8 and 9,respectively. The light chain variable amino acid and nucleic acidsequences of mature m1H7 are provided as SEQ ID NOS:10 and 11,respectively. The heavy chain CDR1, CDR2, and CDR3 amino acid sequencesare provided as SEQ ID NOS:12, 13, and 14, respectively. The light chainCDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOS:15,16, and 17, respectively. Kabat numbering is used throughout in thisExample.

The variable kappa (Vk) of m1H7 belongs to mouse Kabat subgroup 3 whichcorresponds to human Kabat subgroup 1. The variable heavy (Vh) of 1H7belongs to mouse Kabat subgroup 5a which corresponds to human Kabatsubgroup 1 (Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242, 1991). The 15residue CDR-L1 belongs to canonical class 5 (note that the Methionine atposition 33 is usually Leucine in this class), the 7 residue CDR-L2belongs to canonical class 1, the 9 residue CDR-L3 belongs to canonicalclass 1 in Vk (Martin & Thornton, J Mol. Biol. 263:800-15, 1996). The 5residue CDR-H1 belongs to canonical class 1, the 17 residue CDR-H2belongs to canonical class 2 (Martin & Thornton, J Mol. Biol.263:800-15, 1996). The CDR-H3 has no canonical classes, but the 8residue loop probably has a kinked base according to the rules of Shiraiet al., FEBS Lett. 455:188-97 (1999).

The residues at the interface between the Vk and Vh domains are the onescommonly found, except that F46 in the kappa chain is usually a Leucine.This makes this position a candidate t for backmutation. A search wasmade over the protein sequences in the PDB database (Deshpande et al.,Nucleic Acids Res. 33: D233-7, 2005) to find structures which wouldprovide a rough structural model of 1H7. The 0.5 B anti-HIV antibody hasgood overall sequence similarity to 1H7 Vk, retaining the same canonicalstructures for the loops. The NMR structure of the 0.5 B anti-HIVantibody (pdb code 1QNZ; Tugarinov et al., Structure 8:385-95, 2000) wasused for the Vk structure in the modeling. Theanti-alpha-(2→8)-polysialic acid antibody has good overall sequencesimilarity to 1H7 Vh structure. It also has a CDR-H3 of a similar lengthwith a kinked base. The structure of the anti-alpha-(2→8)-polysialicacid antibody (1PLG; Evans et al., Biochemistry 34:6737-44, 1995) hasreasonable resolution (2.8 A), and was used for the Vh structure in themodeling. In addition, CDRs-H1 and H2 of the anti-alpha-(2→8)-polysialicacid antibody have the same canonical structures as 1H7 Vh.DeepView/Swiss-PdvViewer 3.7 (SP5) (Guex & Peitsch, Electrophoresis 18:2714-2723, 1997) was used to model a rough structure of 1H7fv.

A search of the non-redundant protein sequence database from NCBIallowed selection of suitable human frameworks into which to graft themurine CDRs. For Vk, a human kappa light chain with NCBI accession codeAAY33358 (GI:63102905; SEQ ID NO:43) (Kramer et al., Eur J. Immunol.35:2131-45, 2005) was chosen. This has the same canonical classes forCDR-L2 and L3, and belongs to human kappa germline A30, a member ofKabat human kappa subgroup 1. AAY33358 has a sequence identity of 65.4%in the light chain variable region framework to murine 1H7 light chain.For Vh, human Ig heavy chain BAC02037 (GI:21670055; SEQ ID NO:42) waschosen, belonging to human heavy germline VH1-18. It is a member ofKabat human heavy subgroup 1. It shares the canonical form of 1H7CDR-H1and H2, and H3 is 8 residues long with a predicted kinked base. BAC02037has a sequence identity of 65.8% in the variable region framework tomurine 1H7 heavy chain. Humanized 1H7 heavy and light chain variablesequences having no backmutations or CDR mutations are provided as SEQID NOS:44-45.

Four humanized light chain variable regions variants and five humanizedheavy chain variable region variants were constructed containingdifferent permutations of the above substitutions (Hu1H7VLv1-v4; SEQ IDNOs:32-39, and Hu1H7VHv1-v5; SEQ ID NOs:18-27 respectively) (FIGS. 1-2and Table 1). SEQ ID NOs. 19, 21, 23, 25, 27, 33, 35, 37, and 39 includebackmutations as shown in Table 1. In addition, two humanized heavychains (SEQ ID NOs: 25, 27) include C→S mutation at position H97 (Kabatnumbering) of heavy chain CDR3 (FIGS. 1-2 and Table 1). The amino acidsat L46, L49, L83, H11, H28, H38, H48, H67, H69, H71, H91, and H97 inHu1H7VLv1-v4 and Hu1H7VHv1-v5 are listed in Table 2.

The H3L3 variant (hu1H7VHv3 (SEQ ID NO:23)-Hu1H7VLv3 (SEQ ID NO:37) wasfound to give the lowest dissociation constant (highest associationconstant), the same as the mouse 1H7 within the SEM.

TABLE 1 V_(H), V_(L) backmutations, and CDR mutations donor frameworkCDR V_(H) variant V_(H) exon acceptor sequence residues mutationsHu1H7VHv1 NCBI accession code H11, H28, H38, H48, (SEQ ID NO: 19)BAC02037 H67, H69, H71, H91 Hu1H7VHv2 NCBI accession codeH28, H48, H67, H69, (SEQ ID NO: 21) BAC02037 H71, H91 (SEQ ID NO: 42)Hu1H7VHv3 NCBI accession code H67, H71 (SEQ ID NO: 23) BAC02037(SEQ ID NO: 42) Hu1H7VHv4 NCBI accession code H28, H48, H67, H69, H97(SEQ ID NO: 25) BAC02037 H71, H91 (SEQ ID NO: 42) Hu1H7VHv5NCBI accession code H67, H71 H97 (SEQ ID NO: 27) BAC02037(SEQ ID NO: 42) donor framework VL variant VL exon acceptor sequenceresidues Hu1H7VLv1 NCBI accession code L46, L49, L83 (SEQ ID NO: 33)AAY33358 (SEQ ID NO: 43) Hu1H7VLv2 NCBI accession code L46, L83(SEQ ID NO: 35) AAY33358 (SEQ ID NO: 43) Hu1H7VLv3 NCBI accession codeL46, L49 (SEQ ID NO: 37) AAY33358 (SEQ ID NO: 43) Hu1H7VLv4NCBI accession code L46 (SEQ ID NO: 39) AAY33358 (SEQ ID NO: 43)

TABLE 2 Kabat numbering of some framework residues for backmutation andCDR mutations in humanized 1H7 antibodies AAY33358 BAC02037 light heavyMouse Hu1H7 Hu1H7 Hu1H7 Hu1H7 Hu1H7 Hu1H7 Hu1H7 Hu1H7 Hu1H7 chain chain1H7 VH1 VH2 VH3 VH4 VH5 VL1 VL2 VL3 VL4 H11 — V L L V V V V — — — — H28T S S S T S T — — — — H38 — R K K R R R R — — — — H48 — M I I I M I M —— — — H67 — V A A A A A A — — — — H69 — M L L L M L M — — — — H71 — T AA A A A A — — — — H91 — Y F F F Y F Y — — — — H97 — G C C C C S S — — —— L46 L — F — — — — — F F F F L49 Y — C — — — — — C Y C Y L83 F — A — —— — — A A F F

The rationales for selection of the above positions as candidates forsubstitution are as follows.

L46F (here as elsewhere for framework backmutations, the first mentionedresidue is the human residue and the second the mouse residue): Thisposition is a Vk/Vh interface residues.

Y49C: Cysteine at this position is unusual in either mouse or humansequence. Occupying a position in the center of the antigen-bindingsite, this residue may bind antigen, or maintain the conformation of theloops.

F83A: In human framework, this position is occupied by phenylalanine, alarger amino acid. Therefore, A83 would be unusual in human frameworks.In the humanized antibody, the constant domain will be human not mouse,so the human F83 would be usual. However, this position in Vk is inclose proximity to the constant domain and may interfere with packingagainst the constant region. Therefore, it would be interesting tobackmutate it to A to see if there is a difference.

V11L: This position contacts the constant domain and may therefore alterthe topography of the binding site.

T28S: This position contributes to CDR-H1 conformation, but may alsobind antigen. The T→S mutation is a conservative mutation.

R38K: This position lies beneath CDR-H2, interacting with F63 in themodel. The R→K mutation is a conservative mutation.

M48I: This position lies beneath F63 in CDR-H2 in the model. The M→Imutation is a conservative mutation.

V67A: This position lies underneath CDR-H2. The V→A mutation is not aconservative mutation.

M69L: This position lies beneath CDR-H2. The M→L mutation is aconservative mutation.

T71A: This position is a canonical residue for CDR-H2. The T→A mutationis not a conservative mutation.

Y91F: This position is an interface residue interacting with P44 in thelight chain. The Y→F mutation is a conservative mutation.

C97S: This CDR mutation in CDRH3 avoids posttranslational modificationof the cysteine.

>Hu1H7Vκ Version1 (SEQ ID NO: 33) DIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMN WYQQKPGK APKFLIC AASNLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC QQSNEDPFT FGQGTKLEIK  >Hu1H7Vκ Version2(SEQ ID NO: 35) DIQLTQSPSSLSASVGDRVTITC KASQSVDYDGDSYMN WYQQKPGK APKFLIYAASNLES GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC QQSNEDPFT FGQGTKLEIK  >Hu1H7VκVersion3 (SEQ ID NO: 37) DIQLTQSPSSLSASVGDRVTITC KASQSVDYDGDSYMNWYQQKPGK APKFLIC AASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSNEDPFTFGQGTKLEIK  >Hu1H7Vκ Version4 (SEQ ID NO: 39) DIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMN WYQQKPGK APKFLIY AASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSNEDPFT FGQGTKLEIK  >Hu1H7vh Version1(SEQ ID NO: 19) QVQLVQSGAELKKPGASVKVSCKASGYSFT SYYIH WVKQAPGQGL EWIGWIYPGSGNTKYSEKFKG RATLTADTSTSTAYMELRSLRSDD TAVYFCAR DGCYGFAYWGQGTLVTVSS  >Hu1H7vh Version2 (SEQ ID NO: 21)QVQLVQSGAEVKKPGASVKVSCKASGYSFT SYYIH WVRQAPGQGL EWIG WIYPGSGNTKYSEKFKGRATLTADTSTSTAYMELRSLRSDD TAVYFCAR DGCYGFAYWGQGTLVTVSS  >Hu1H7vh Version3 (SEQ ID NO: 23)QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYIH WVRQAPGQGL EWMG WIYPGSGNTKYSEKFKGRATMTADTSTSTAYMELRSLRSDD TAVYYCAR DGCYGFAYWGQGTLVTVSS  >Hu1H7vh Version4 (SEQ ID NO: 25)QVQLVQSGAEVKKPGASVKVSCKASGYSFT SYYIH WVRQAPGQGL EWIG WIYPGSGNTKYSEKFKGRATLTADTSTSTAYMELRSLRSDD TAVYFCAR DGSYGFAY WGQGTLVTVSS >Hu1H7vh Version5(SEQ ID NO: 27) QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYIH WVRQAPGQGL EWMGWIYPGSGNTKYSEKFKG RATMTADTSTSTAYMELRSLRSDD TAVYYCAR DGSYGFAYWGQGTLVTVSS 

Example II Binding Kinetic Analysis of Murine, Chimeric, and Humanized1H7 Antibodies

Binding kinetics of humanized 1H7 antibodies comprising a heavy chainselected from Hu1H7VHv1-5 and a light chain selected from Hu1H7VLv1-4have been characterized.

Biacore full binding kinetic analysis of antibodies were carried outusing Biacore. Detailed binding kinetic parameters (association rate,ka, dissociation rate, kd, and affinity constant, KD) were determinedfor murine 1H7 (FIG. 3A), chimeric 1H7 (FIG. 3B) and humanized 1H7(Hu1H7VHv3-Hu1H7VLv3, Hu1H7VHv3-Hu1H7VLv1, Hu1H7VHv4-Hu1H7VLv1)antibodies (FIG. 3C). Binding kinetic parameters of humanized 1H7, inparticular Hu1H7VHv3-Hu1H7VLv3, are comparable to those of murine 1H7.

TABLE 3 Binding kinetic parameters of murine 1H7, chimeric 1H7 andhumanized 1H7 (Hu1H7VHv3-Hu1H7VLv3, Hu1H7VHv3- Hu1H7VLv1,Hu1H7VHv4-Hu1H7VLv1) ka kd K_(D) Antibody (1/Ms) (1/s) (nM) Murine 1H71.0e6 9.6e−3 9.5 Chimeric 1H7 1.7e6 1.3e−2 7.4 Humanized 1H7 H3L3 1.3e61.1e−2 9.0 (Hu1H7VHv3-Hu1H7VLv3) Humanized 1H7 H3L1 1.1e6 1.3e−2 12(Hu1H7VHv3-Hu1H7VLv1) Humanized 1H7 H4L1 9.7e5 3.3e−2 33.9(Hu1H7VHv4-Hu1H7VLv1) Humanized 1H7 H5L1 8.8e5 5.6e−2 64.0(Hu1H7VHv5-Hu1H7VLv1) Humanized 1H7 H4L4 N/A * N/A * N/A *(Hu1H7VHv4-Hu1H7VLv4) * The binding kinetic parameters could not bedetermined for Hu1H7VHv4-Hu1H7VLv4 at concentrations comparable to thoseused in the analysis for other humanized 1H7 antibodies ormurine/chimeric 1H7.

Binding kinetic of humanized 1H7 antibodies were also measured bybio-layer interferometry (BLI) using a ForteBio Octet QK instrument(ForteBio, Menlo Park, Calif.). Detailed binding kinetic parameters(association rate, apparent ka, dissociation rate, apparent kd, andaffinity constant, apparent KD) were determined for chimeric 1H7 andvarious humanized 1H7 antibodies (Tables 6-7, FIGS. 4A-C). Apparent ka,apparent kd and apparent K_(D) are binding kinetic parameters obtainedusing ForteBio assay formats. These parameters differ from ka, kd andK_(D) measured using Biocore assays due to, e.g., avidity effectsassociated with ForteBio assay formats.

TABLE 4 Binding kinetic parameters of chimeric 1H7 and humanized 1H7(Hu1H7VHv1-Hu1H7VLv1, Hu1H7VHv1-Hu1H7VLv2, Hu1H7VHv1-Hu1H7VLv3,Hu1H7VHv1-Hu1H7VLv4, Hu1H7VHv2- Hu1H7VLv1, Hu1H7VHv2-Hu1H7VLv2) ApparentApparent Apparent Antibody ka (1/Ms) kd (1/s) K_(D) (nM) Chimeric 1H74.7e5 1.1e−6 2.3e−3 Humanized 1H7 H1L1 2.5e5 1.2e−3 4.8(Hu1H7VHv1-Hu1H7VLv1) Humanized 1H7 H1L2 1.6e6 9e−3 5.8(Hu1H7VHv1-Hu1H7VLv2) Humanized 1H7 H1L3 2.2e5 6.4e−4 2.9(Hu1H7VHv1-Hu1H7VLv3) Humanized 1H7 H1L4 1.8e6 5.8e−3 3.3(Hu1H7VHv1-Hu1H7VLv4) Humanized 1H7 H2L1 5.8e5 4.2e−6 7.2e−3(Hu1H7VHv2-Hu1H7VLv1) Humanized 1H7 H2L2 2.3e6 4.6e−3 2(Hu1H7VHv2-Hu1H7VLv2)

TABLE 5 Binding kinetic parameters of chimeric 1H7 and humanized 1H7(Hu1H7VHv4-Hu1H7VLv2, Hu1H7VHv4-Hu1H7VLv3, Hu1H7VHv4-Hu1H7VLv4,Hu1H7VHv5-Hu1H7VLv2, Hu1H7VHv5- Hu1H7VLv3, Hu1H7VHv5-Hu1H7VLv4) ApparentApparent Apparent Antibody ka (1/Ms) kd (1/s) K_(D) (nM) Chimeric 1H78.3e5 8.7e−4 1.0 Humanized 1H7 H4L2 1.2e6 2.1e−3 1.7(Hu1H7VHv4-Hu1H7VLv2) Humanized 1H7 H4L3 7.4e5 2.0e−3 2.7(Hu1H7VHv4-Hu1H7VLv3) Humanized 1H7 H4L4 5.2e5 2.3e−3 4.4(Hu1H7VHv4-Hu1H7VLv4) Humanized 1H7 H5L2 5.2e5 2.1e−3 4.0(Hu1H7VHv5-Hu1H7VLv2) Humanized 1H7 H5L3 7.6e5 2.0e−3 2.6(Hu1H7VHv5-Hu1H7VLv3) Humanized 1H7 H5L4 1.6e6 1.9e−3 1.2(Hu1H7VHv5-Hu1H7VLv4)

Humanized 1H7 antibodies, in particular Hu1H7VHv3-Hu1H7VLv3 andHu1H7VHv3-Hu1H7VLv1, exhibited staining patterns of various regions(e.g., striatum, pyramidal cell layer, cortex, substantia nigra) oftransgenic or non-transgenic mouse brain similar to those of murine 1H7.

Example II Passive Immunization with α-Synuclein Antibodies

The goal of this experiment is to determine effectiveness of α-synucleinantibodies in in vitro and in vivo studies as well as behavioral assays.We used α-synuclein transgenic (Line 61), α-synuclein knockout andwildtype female mice, 3-4 months old at initiation and n=14/group.Antibodies tested included 9E4 (IgG1, epitope: amino acids 118-126 ofalpha synuclein), 5C1 (IgG1, epitope: amino acids 118-126 of alphasynuclein, c-linker), 5D12, IgG2 (SN118-126), 1H7, IgG1 (SN 91-99) andan IgG1 control antibody 27-1. Mice received a dosage of 10 mg/kg over a5 month period, for a total of 21 injections. In addition, the animalswere injected with lentivirus (LV) expressing human α-synuclein (wt) byunilateral introduction of human α-synuclein (wt) into the hippocampus.

Readout antibodies include those from Chemicon (epitope: full-lengthalpha synuclein), Millipore (epitope: full-length alpha synuclein), andNeotope, ELADW 105 (epitope: amino acids 121-124 of full-length alphasynuclein).

Endpoints: Antibody titers were measured during the in life phase.Behavioral assays include Morris Water Maze test (MWW) and horizontalbeam test. The round beam test is a test of motor balance, coordinationand gait conducted using two beams of varying diameter. Beam A is thelarger diameter (easier, considered the training beam) and Beam D is thesmaller diameter (more difficult, considered the testing beam). Data ispresented as “errors” (number of slips/10 cm) and “speed” (time taken totravel 10 cm/sec). Water maze performance was carried out at weeks 10and termination. The following neuropathology measurements were taken:alpha synuclein aggregation, synaptophysin, and MAP2. The followingbiochemistry measurements were taken: alpha synuclein, PSD95,synaptophysin. Selected multilabeling and confocal labeling were carriedout using synaptic, neuronal and glial markers.

The results showed that all antibodies, except 5D12, producedsignificant reduction in α-syn accumulation and preservation of synapticand dendritic densities, as well as positive outcomes in MWMperformance. The 9E4 antibody is effective in in vitro and in vivostudies as well as behavioral assays. Readouts indicate antibody mayreduce neuritic/axonal alpha synuclein aggregates.

Behavioral Results: The 1H7 antibody (as well as 9E4 and 5C1 antibodies)improved water maze performance in α-synuclein transgenic mice, whereas5D12 did not. The 9E4 and 1H7 antibodies improved performance on thebeam test as measured both by speed and errors, whereas the 5D12 and 5C1antibodies did not (FIG. 6).

Neuropathology Results: The 9E4, 1H7 and 5C1 antibodies reducedELADW-105 positive neuritic dystrophy, whereas the 5D12 antibody didnot. In alpha synuclein transgenic mice, the 9E4 antibody reduced thearea of neuropil by 43% in neocortex and by 40% in basal ganglia ascompared to control. The 9E4 antibody also preserved synaptophysin andMAP2 in neocortex and basal ganglia.

Deposit

The following monoclonal antibody-producing cell lines have beendeposited under the provisions of the Budapest Treaty with the AmericanType Culture Collection (ATCC, P.O. Box 1549, Manassas, Va. 20108) onthe dates indicated:

Mono- Acces- clonal Epitope/ Iso- Date of sion antibody Cell LineSpecificity type Deposit No. 1H7 JH17.1H7.4.24.34 alpha- IgG1 Feb. 26,PTA-8220 synuclein 2007 residues 91-99

It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made thereto without departing from thespirit or scope of the appended claims. Unless otherwise apparent fromthe context, any step, feature, embodiment, or aspect can be used incombination with any other. All publications and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

What is claimed is:
 1. A nucleic acid encoding a mature heavy chainvariable region and/or a mature light chain variable region of anantibody comprising a mature heavy chain variable region, whereinposition H97 by Kabat numbering is C, M, S or T and other residues ofthe three Kabat CDRs are as defined by SEQ ID NO:44, and the heavy chainvariable region is at least 90% identical to SEQ ID NO:44, and a maturelight chain variable region comprising the three Kabat CDRs of SEQ IDNO:45, and being at least 90% identical to SEQ ID NO:45, wherein theantibody specifically binds to human alpha synuclein.
 2. The nucleicacid of claim 1, wherein the amino acid sequence of the mature heavychain variable region is SEQ ID NO:44 and the amino acid sequence of themature light chain variable region is SEQ ID NO:45 except provided thatposition L46 (Kabat numbering) is occupied by L or F, and/or positionL49 (Kabat numbering) is occupied by Y or C, and/or position L83 (Kabatnumbering) is occupied by F or A, and/or position H11 (Kabat numbering)is occupied by V or L, and/or position H28 (Kabat numbering) is occupiedby T or S, and/or position H38 (Kabat numbering) is occupied by R or K,and/or position H48 (Kabat numbering) is occupied by M or I, and/orposition H67 (Kabat numbering) is occupied by V or A, and/or positionH69 (Kabat numbering) is occupied by M or L, and/or position H71 (Kabatnumbering) is occupied by T or A, and/or position H91 (Kabat numbering)is occupied by Y or F, and/or H97; (Kabat numbering) is occupied by C orS, and wherein the antibody specifically binds to human alpha synuclein.3. The nucleic acid of claim 1, having a heavy chain sequence comprisingSEQ ID NO:22 and/or a light chain sequence comprising SEQ ID NO:36. 4.The nucleic acid of claim 1, having a heavy chain sequence comprisingSEQ ID NO:22 and a light chain sequence comprising SEQ ID NO:36.
 5. Thenucleic acid of claim 1, wherein the mature heavy chain variable regionhas an amino acid sequence designated SEQ ID NO:23 and the mature lightchain variable region has an amino acid sequence designated SEQ IDNO:37.
 6. The nucleic acid of claim 5, wherein the mature heavy chainvariable region is fused to a heavy chain constant region having theamino acid sequence designated SEQ ID NO:52, wherein the C-terminallysine residue is optionally omitted and the mature light chain constantregion is fused to a light chain constant region having the amino acidsequence designated SEQ ID NO:49.
 7. The nucleic acid of claim 1 havinga sequence comprising any one of SEQ ID NO: 18, 20, 22, 24, 26, 32, 34,36, or
 38. 8. A host cell comprising a vector comprising the nucleicacid of claim 1 or
 7. 9. A method of producing an antibody, comprisingculturing cells transformed with nucleic acids encoding the heavy andlight chains of the antibody, so that the cells secrete the antibody;and purifying the antibody from cell culture media; wherein the antibodyis defined by claim 1 or 7.